Rat pheochromocytoma PC12 cells and bovine adrenal chromaffin cells are used to study the mechanism of secretion and its regulation by Ca2+ and GTP-binding proteins. We have found that modification of chromaffin cells with pertussis toxin causes a 40-50% decrease in the amount of cytoskeletal actin. This depolarization of F-actin may account for the increased secretory activity of pertussis toxin-modified chromaffin cells. This decrease in F-actin appears to be independent of changes in known second messengers and may result from a direct interaction of a G-protein with the cytoskeleton. To our knowledge this is the first report that modification with pertussis toxin can reduce the level of F-actin in an unstimulated cell. We have isolated a cytosolic protein which appears to play an important role in regulated exocytosis. Incubation of digitonin- permeabilized bovine chromaffin cells in the absence of Ca2+ results in a loss of both cytosolic proteins and Ca2+-dependent secretion. Addition of these leaked proteins prevents this loss of secretory activity. We have purified a protein from an extract of bovine adrenal medulla which can partially prevent this loss of Ca2+-dependent secretion. Antibody against this protein inhibited the ability of leaked chromaffin cell proteins to prevent the loss of Ca2+-dependent secretion. Sequence analysis showed it to be very similar if not identical to bovine brain 14-3-3 protein, a protein not previously thought to be involved in exocytosis. Secretion of norepinephrine by digitonin permeabilized PC12 cells can be stimulated by the addition of GTP7S in the absence of Ca2+. We are continuing to try to determine the mechanism by which GTP7S induces secretion in these cells and are examining the possible role of nucleoside diphosphate kinase in this response.